Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. Starch is the main source of energy for a wide variety of living organisms. Amylases are classified according to their enzymatic action pattern.
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Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.
The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. Starch is the main source of energy for a wide variety of living organisms. Amylases are classified according to their enzymatic action pattern.
Glucoamylases EC 3. Pullulanases EC 3. Several groups of starch-hydrolyzing enzymes are known to harbor more than single enzyme activity. One group of these, maltogenic amylases MAases; 1 EC 3. Different sugar molecules, including glucose, fructose, maltose, and cellobiose, can serve as acceptors for the transglycosylation. The property, if not all, is shared by other amylolytic enzymes with different names, including neopullulanases NPases; EC 3. Unlike other starch-hydrolyzing enzymes, the three groups of the enzymes are intracellular enzymes.
In Klebsiella oxytoca , the open reading frames for CDase and an extracellular enzyme CGTase are clustered on the chromosomal DNA together with genes coding for products homologous to the maltose and linear maltodextrin uptake system The finding suggested a starch degradation pathway where CDase is involved in the intracellular degradation of CDs that are generated and transported into the cell by CGTase and by a specific uptake system, respectively. However, structural information of MAases has been lacking.
We have determined the structure of maltogenic amylase from a Thermus strain ThMA. Initial effort was put into the structure determination of Bacillus stearothermophilus maltogenic amylase BSMA whose crystallization condition we have reported We suffered because of fragility of the crystals, especially in the presence of a heavy metal compound, and subsequently switched to the crystallization of ThMA which led to the structure determination of the both enzymes.
Gene cloning and overproduction of ThMA was described recently 5. The cryoprotectant solution contained 0. The translation functions were calculated with the highest peak with a correlation of 7.
The search found the highest peak with a correlation of The increase in the correlation coefficient suggested that the peak was a correct solution. However, efforts to find the position of the second molecule after fixing the position of the first solution did not yield a promising solution. The first solution was rotated according to the noncrystallographic symmetry NCS , and a translation search along the xy plane was performed, which was followed by a translation search along the z -axis to correlate the relative z -positions of the two solutions.
The calculations generated the final solutions with an R -factor of When examined on a graphics computer, the two molecules showed no overlap with symmetry-related molecules. Furthermore, the phase derived from MR solutions identified a holmium position in a Fourier difference map which coincides with the position located from 3.
The successful MR demonstrates that initial phase information can be derived using available structures coupled with a correct prediction of the tertiary folding pattern of an interested protein. It was not at all straightforward to solve the structure by using the phase information derived from MR, and additional phase information was obtained from three heavy atom derivative crystals Table I by MIR multiple isomorphous replacement method. Heavy atom binding sites were identified by Fourier difference analysis using the phases derived from MR.
The heavy atom positions were used to calculate MIR phases. The MIR phases with the three derivative data had a mean figure of merit of 0. Some parts of the partial model inconsistent with the MIR map were further truncated. The 2-fold NCS restraints were maintained until the last refinement. The final model at 2. The model does not contain highly disordered residues —, —, and — for which electron densities are lacking.
Contrary to our earlier prediction that the asymmetric unit of BSMA crystals would contain three to four molecules 13 , only one dimer is present with a high solvent content of The values of the two variables, absorbances at nm versus radial positions were obtained.
The partial specific volume of ThMA and solvent density were calculated as described by Zamyatnin The central region exhibits a low degree of sequence homology, but the three catalytic residues, Asp, Glu, and Asp ThMA numbering are invariant.
More than two-thirds of the excluded surface is hydrophobic surface. An ultracentrifugation analysis further evidenced the dimerization of ThMA in solution as shown in Fig. BSMA exhibits the same chromatographic profile and is also dimeric with tertiary and quaternary structures nearly identical to those of ThMA.
Therefore, it is firmly established that MAases are dimeric in solution. Two molecules of TVAII were also contained in the asymmetric unit of the crystals of the enzyme space group P 2 1 2 1 2 1. The two molecules exhibit the same molecular contacts as the ThMA dimer does. Therefore, the substrate profile and catalytic property of these enzymes would be best explained by the active site configuration in the dimeric state.
CDase O , and A. The secondary structure assignment and numbering at the top of the alignment correspond to ThMA. Amino acids that are not conserved, compared with the ThMA sequence, are lightly shaded. The black boxes represent the catalytic residues. Glu is indicated by an asterisk whose substitution with histidine severely affects the transglycosylation activity of ThMA.
Every other 20th residue positions are numbered. The two subunits, related by the molecular 2-fold axis lying on the figure, are labeled with different colors.
The docking of the substrate molecule did not require reorientation of the protein amino acid side chains and change in torsion angles of substrate. The proteins were eluted with 50 m m phosphate buffer pH 7. The flow rate was 0. The inset shows the line fitting of the elution time versus logarithm of the molecular weight of the size markers.
The concentration of ThMA was 0. The equilibrium was attained in 40 h. The concentration distribution of the protein as a function of the square of the radial position is shown. The partial specific volume of ThMA was calculated as 0. The solid line indicates the calculated curve for dimeric species. R 2 , the coefficient of determination, indicates an excellent fit to an ideal dimeric species model. The consequence of the dimer formation of ThMA is striking. The groove must be the active site cleft because the three invariant catalytic residues are located at the bottom of the groove.
They are found in the same relative position in space as the corresponding residues in TAKA-amylase A. The shape of the active site cleft of ThMA explains much slower hydrolysis of starch than CDs by the enzyme.
The ring structures of CDs should be narrower in width than that of starch segment amylose , which assumes a coiled helical structure composed of six, seven, or eight glucose residues per turn of the helix in aqueous solution The program allows a successful docking only when an electrostatic and geometric complementarity is accomplished between a host and a guest molecule.
The catalytic residues of ThMA would be reached only by the disordered part of starch. Extra sugar-binding space at the active site of ThMA. The docking did not require reorientation of the protein side chains.
The active site residues Asp, Glu, Asp plus Glu, identified as important for transglycosylation, are labeled. All oxygen atoms are in red. Unlike CGTase, ThMA mainly cleaves glycosidic bonds at the beginning of the enzyme reaction, and transglycosylation products are detected after a time lag when cleavage products are accumulated. The observation raises a possibility that the transglycosylation by ThMA is the reverse reaction of the hydrolysis driven by a high concentration of small oligosaccharide products.
We established that it is not the case for ThMA. PTS can be recovered in pure form from an enzyme reaction mixture. Therefore, the transglycosylation reaction by ThMA is concomittant with the hydrolysis of glycosidic bond. Given this mechanism and the fact that the transglycosylation reaction requires a high concentration of an acceptor sugar, it is reasonable to think that the acceptor molecule competes with a water molecule at the active site for attacking the glycosyl-enzyme intermediate.
The assumption requires a sugar-binding site adjacent to the main substrate-binding site. A corresponding space is not found in the smaller amylases. The size of the space appears to accommodate a disaccharide such as maltose rather easily, as a maltose molecule can be successfully docked into the space Fig. In this model, the C4-OH group of a maltose at the nonreducing end is 3. There is some degree of freedom to rotate and translate the model without steric clash.
A mono- or disaccharide occupying the space could serve as an acceptor molecule to compete with a water molecule for attacking enzyme-substrate intermediate catalyzed by the same catalytic armory as shown in Fig. In the extra sugar-binding space, an acceptor sugar molecule may be able to position either the C3-, C4-, or C6-OH group in a proper orientation for nucleophilic attack of the glycosyl-enzyme intermediate.
The idea of extra sugar-binding space playing an important role in the transglycosylation explains the effect of Glu mutation. The residue is at hydrogen bonding distance from the modeled maltose molecule. In the context of our proposal, Glu appears to play an important role in the binding of small oligosaccharide acceptors.
Schematic drawings of products from reaction of acarbose with ThMA. Acarbose is hydrolyzed to PTS and glucose. Three different products can be generated by transglycosylation of PTS to glucose. Proposed mechanism for competition of transglycosylation and hydrolysis reaction at the active site of ThMA. A proposal for a double-displacement reaction is followed. The third conserved residue Asp, which may play a role in raising the p K a of Glu 30 , is not drawn.
Bake better bread with enzymes (benefits of Maltogenic Amylase)
A maltogenic amylase MAG1 from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli , purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases.
Maltose units are successively removed from the non-reducing end of the polymer chain until the molecule is completely degraded or, in the case of amylopectin, a branch point is reached. The powdered form contains sodium chloride as a stabilizer, the granulated form contains sodium chloride and wheat grits, and the liquid form contains sodium chloride and sucrose. The reaction is stopped by raising the pH of the reaction medium to about One enzyme unit Maltogenic Amylase Unit, MAU is defined as the amount of enzyme which under standard assay conditions cleaves 1 m mol of maltotriose per minute.
This is a guest post by Morgan Walker Clarke. Morgan is from Dallas, Texas, works in the hospitality industry and has an interest for food and science that he loves to share with us! Using enzymes in baking is not a new concept. Bakers have been using enzyme reactions without even knowing it since the beginning of bread making. Enzymes are found naturally in flour and other ingredients, but not always in large amounts. The amounts of enzymes can also vary from one batch of flour to the next, nor are the natural enzymatic reactions uniform.